Lampbrush chromosome protocols

How to make preparations of Lampbrush
Chromosomes

Because of their great length and delicate
form , lampbrush chromosomes (LBCs) are difficult to study in
sections of the oocyte nucleus or "germinal vesicle (GV)".
This inherent structural problem was already recognized by Rückert,
who isolated and examined intact but fixed GVs from shark oocytes
(Rückert, 1892). Somewhat
later Duryee (1937; 1941;
1950) made a major advance by showing that GVs could be removed
from living oocytes and the nuclear envelope removed to allow
direct access to the unfixed chromosomes and nucleoli. Duryee
stressed the importance of Ca++-free solutions for
maintaining lifelike chromosome structure, and all subsequent
protocols have been based on his crucial observation. Finally,
attachment of the chromosomes and other nuclear organelles to
a glass slide is essential for conventional or immunofluorescent
staining and particularly for in situ hybridization. All recent
protocols include a centrifugation step to assure attachment of
the GV contents to the slide.

Freshly isolated and unfixed LBCs can only
be examined at high magnification if the chromosomes are dissected
from the GV into a chamber the bottom of which consists of a coverglass
and then observed with an inverted phase contrast optical system,
looking up through the bottom of the chamber. This technical approach
was devised by J.G. Gall in
1954 and opened the way for all modern experimental studies of
these remarkable objects.

Four protocols for making and examining preparations
of LBCs are available here. "Getting
Started" will introduce you to
some of the very basic aspects of LBC technology. It is based
on a successful undergraduate laboratory teaching exercise. The
other 3 protocols explain how to work with LBCs from urodeles (mainly newts of the genera Triturus and Notophthalmus),
Xenopus laevis, and birds (with special regard to the chicken, Gallus).

Each
protocol can be downloaded and printed out as a WORD document,
for your convenience.

Have lots of fun!

Other specialist protocols for experimental approaches to lampbrushology
will be added later to this site.